Differences in various phenotypes of humans, including disease, are known to be derived from the differences in DNA nucleotide sequence in an individual genome. This difference is called single nucleotide polymorphism (SNP). SNPs are found widely in human genome of about 3 billion base pairs and the total number of SNPs is not less than 3 millions. Thus, SNPs can be the DNA markers having exceptionally higher density that known DNA markers such as RFLP (restriction fragment length polymorphism) and STR (microsatellite), which are conventional DNA markers. Therefore, high-precision analysis, which has been impossible using conventional DNA markers, is possible by using SNPs, and it is hoped that SNP can be applied to detection of disease genes, determination of disease sensitivity, and development of pharmaceutical.
Known methods for detecting DNA polymorphism including SNP are as follows:
(1) PCR-SSCP (Single-strand Conformation Polymorphism)
When double strand DNA fragment amplified by PCR is separated by electrophoresis, its electrophoretic mobility is proportionate to the size of the DNA molecule. When single strand DNA molecule is electrophoresed with gel including denaturant, its electrophoretic mobility is proportional to the size of the molecule. When it is electrophoresed without denaturant, single strand DNA forms folded structure (higher order structure) due to intramolecular interaction. Thus, electrophoretic mobility of the DNA forming such folded structure varies depending on its structure. In the PCR-SSCP method, double strand DNA amplified by PCR is denatured by heat or alkaline treatment. Then, the single strand DNA obtained is electrophoresed with polyacrylamide gel without denaturant. The single strand DNA is folded due to intramolecular interaction and forms higher order structure. The interaction of the folded structure can vary depending on the difference of nucleotide. The difference can be detected by staining the DNA fragment electrophoresed. Ethidium bromide widely used for the detection of DNA stains double strand DNA, but it does not stain single strand DNA usually. Therefore, silver staining is applied in this method.
However, there is a problem with this technique because the region for searching is from 200 to 300 bp and the detection fails when the region is not more than that.
(2) Direct Sequencing
A direct sequencing method is the method in which nucleotide sequence is determined directly using DNA amplified by PCR as a template without subcloning with vector. This method can remove misreading which is a drawback of PCR. As misreading of PCR is said to occur once over 400 bp, one nucleotide substitution per 400 nucleotides should be detected after subcloning. However, because errors in nucleotide sequence introduced randomly are diluted into 1/400 on an average, they mostly can be ignored in the direct sequencing method. In this method, after amplifying DNA strand by primary PCR, secondary PCR called asymmetric PCR that amplifies single strand DNA is performed. Then, the nucleotide sequence is generally determined using dideoxy method. This secondary PCR amplifies single strand DNA by conducting PCR using a pair of primers in which limited amount of one primer is used (1:10˜1:100, usually).
Because this method needs to sequence genes one after another, the probability to find the mutation is low. Also, the mutation can be missed because of the problem of sequencer.
(3) IGCR (In-gel Competitive Reassociation) Method
There is the method using competitive reassociation of DNA in gel as a method for cloning DNA having slightly different structure. Using reference DNA dephosphorylated, DNA is denatured in electrophoresis gel, reassociated, recovered, and cloned so that the target DNA fragment having changed structure is selectively concentrated.
However, this technique is searching for longer region than that in PCR-SSCP method. It has problems in which, for example, it cannot detect repetitive sequence because of its mechanism.
Therefore, it was hoped to develop efficient and certain method for detecting polymorphism that does not require long DNA region for searching.